Endoplasmic reticulum (ER) chaperones are multifunctional proteins involved in a variety of biological processes such as protein folding and quality control in the ER (Hebert et al., 1995; Zhang et al., 1997; Braakman & van Anken, 2000; High et al., 2000; Bedard et al., 2005; Benyair et al., 2011; Braakman & Bulleid, 2011), unfolded protein response (Spear & Ng, 2001; Ma & Hendershot, 2004; Malhotra & Kaufman, 2007; Groenendyk et al., 2010; Chakrabarti et al., 2011), MHC class I antigen processing (Maffei et al., 1997; Nicchitta & Reed, 2000; Zhang & Williams, 2006; Wearsch & Cresswell, 2009), as well as other important functions these proteins play outside of the ER (Panayi & Corrigall, 2006; Gonzalez-Gronow et al. 2009; Gold et al., 2010; Ni et al., 2011; Peters & Raghavan, 2011; Turano et al., 2011). The role of ER chaperones in various human diseases seems especially important. There are growing amounts of data demonstrating involvement of particular ER chaperones in many pathological processes. For example, ER chaperone GRP78/BiP appears to be involved in cancer progression (Li & Lee, 2006; Lee, 2007; Luo & Lee, 2012), autoimmune inflammation and tissue damage (Panayi & Corrigall, 2006; Morito & Nagata, 2012) and rheumatoid arthritis (Corrigall et al., 2001; Yoo et al., 2012). Another ER chaperone calreticulin plays an important role in activating the anti-tumor response needed in chemotherapy or various other cancer treatment strategies (Chaput et al., 2007; Obeid et al., 2007; Wemeau et al., 2010) and is also associated with the healing processes of cutaneous wounds (Nanney et al., 2008). Other ER chaperones have also been implicated in disease related processes, such as prion diseases in the case of chaperone GRP58/ERp57 (Hetz et al., 2005). These recent findings suggest possible application of ER chaperones in therapeutic trials and development of new pharmaceuticals. Therefore, the growing demand of human ER chaperone protein products could be expected in the near future.
Native human ER chaperone proteins can be purified from various tissues, e.g. calreticulin has been purified from human placenta (Houen & Koch, 1994), however human tissues are not a sufficient source of these proteins for large scale clinical trials. The recombinant protein expression technologies should be considered for efficient and safe production of these proteins. Furthermore, it is desirable that the recombinant proteins for clinical trials should correspond to native analogs insofar as possible.
Currently most recombinant human ER chaperones are produced in bacterial host Escherichia coli (Rokeach et al., 1991; Baksh et al., 1992; Antoniou et al., 2002) and such products are commercially available (Abcam products ab78432, ab91577 and ab92937, 2012; StressMarq product SPR-119B, 2012; USBiological products B1770-01, C1036-02L1 and E2291-75E, 2012). However, E. coli and other prokaryotes do not possess the ER, Golgi apparatus and other organelles of the eukaryotic secretion pathway, therefore it is uncertain that human ER proteins produced in bacteria will be correctly folded and possess all the same functions as the native protein analogues. Yeast is an attractive host for the production of the ER chaperones and other complex secreted human proteins, because this unicellular eukaryotic microorganism has eukaryotic features including a secretory pathway leading to correct protein processing and post-translational modifications (Mattanovich et al., 2012). Many attempts have being made to generate recombinant secreted human proteins in yeast (Damasceno et al., 2012; Hou et al., 2012) as such expression system facilitates purification and downstream processing of the protein product and the secreted proteins often are biologically active. Regarding generation of the secreted human ER chaperone proteins in yeast, several techniques may be used. All these approaches include use of the conventional yeast protein secretion signal fused to the sequence of processed mature human ER protein. The protein product generated in this way has several non-native amino acids on the N-terminus and the effect of this manipulation to biological activity of the prepared proteins is unclear. The only known example of yeast-expressed secreted full-length recombinant mammalian ER chaperone described in the literature so far is generation of recombinant rabbit calreticulin in yeast Pichia pastoris (Andrin et al., 2000).